Ruedi Aebersold and Hui Zhang of the Institute for Systems Biology outline a method in US 2010/0222233 to address two current limitations in protein analysis: the inability of microarrays to reliably quantify protein amount and the inability of mass spectrometry to detect proteins of low abundance. The ISB approach is essentially to use both methods in sequence, with a microarray step preceding a MALDI-MS step. The use of an initial array step to retain peptides of interest (including fragments of low abundance proteins) enables subsequent MS analysis of low abundance proteins, and detection settings can be tuned for specific protein fragments to further enhance sensitivity. As outlined in the patent, the challenge in quantification with microarrays is that target protein and array element pairs will each have different optimal binding conditions, and target proteins will have solubility variations that are also dependent on solution conditions. However, use of heavy isotope peptide standards of known concentration solves this problem: Loss of target peptide during washing steps on the microarray will be accompanied by similar loss of its heavy-isotope peptide-standard counterpart, but the exact starting quantities of the heavy-isotope peptides are known, so the ratios of resulting MS signals (heavy/light) will yield the quantities of target peptides.

The approach outlined in the patent is an advance to the state of the art, but remains limited as a proteomics tool since it only works for previously characterized proteins. Basically, you have to know what you’re looking for already. It’s worth pointing out that the idea of separating incoming molecules prior to mass spectrometry analysis is not new (e.g., GC-MS instruments were in widespread use beginning around 20 years ago). However, the protein chip’s high degree of specificity and high-throughput nature – when used in combination with mass spectrometry – is new.

Reveo is developing an ambitious technology to stretch out and deposit taut DNA on conductive surfaces for electronic base detection using one or more STM tips and tunneling current measurements. The linearization and deposition of nucleic acid sequences will likely be done using molecular combing. Reveo’s approach requires atomically flat and positively charged substrate surfaces (e.g. self-assembled monolayers on gold substrates or treated graphite substrates). In addition to molecular combing, Reveo has proposed other methods to linearize DNA, including electrophoretic and hydrodynamic stretching and transfer printing[1]. Furthermore, Reveo has proposed to develop STM tips that are knife-edge shaped, where the smallest dimension is nanoscale[2, 3].

Schematic of Reveo's DNA sequencing method from US 2009/0121133

Much like with IBM/Roche’s proposed DNA transistor, Reveo’s competitive advantage is largely based on the potential cost reduction associated with avoiding labels and the possibility of exceptionally long read lengths. In principle, there’s not much difference between Reveo’s technology and that of IBM’s DNA transistor: both stretch and confine DNA to allow for tunneling current measurement of individual bases (albeit in different geometrical arrangements). IBM’s approach will likely be more reproducible and offer a higher degree of control over local DNA segment position. It’s possible that Reveo’s immobilization approach will reduce smaller-scale configurational rearrangement and motion compared to IBM’s approach, but Reveo’s method will likely be undermined by irregularities in DNA deposition. Reveo’s knife-edge tip design is intriguing, as it would avoid cumbersome issues of probe tip and DNA backbone alignment. Interestingly, even with nanoscale knife-edge tips it appears that individual, isolated DNA molecules would be required as a starting point for analysis, as simultaneous analysis of multiple strands would only be feasible if local DNA contour were uniform along an entire chain length (thus permitting the deconvolution of signals from multiple DNA strands given each individual strand would generation a periodic signal).

Outside of its entry into the Archon X Prize for Genomics, little is known about Reveo’s efforts in DNA sequencing. The company was founded in 1991, has spun out a number of companies, and holds over 300 patents across multiple technology and product areas[4]. Furthermore, although Reveo announced a partnership with the University of Washington (Babak Parviz’s lab) in 2006[5], a 2008 Nature Methods article describing Reveo’s technology did not reference the University of Washington[3]. The University of Washington was awarded a $1.5 million grant in 2006 to develop this technology and Reveo has cited this as its own funding [6, 2]. US patent application 2009/121133 described above lists Parviz as the inventor and University of Washington as the assignee; it’s quite possible that Reveo has priority rights to this and related, future University of Washington patents.

Mobious Biosystems was founded in 1999. It is developing instruments to detect single polymerase conformation or mass changes during the sequencing-by-synthesis process, using physical methods not dependent on the use of fluorophores. The start-up does not appear to have developed anything at this time that will significantly impact the third generation sequencing market. It does appear to have made progress on PCR and hybridization array technologies[1]. It is important, however, to note the impressive range of sequencing ideas and corresponding patents generated by founder Daniel Densham and the small company (see below). Conversely, it’s important to recognize that nearly all of Mobious’ technologies either currently are (or initially were) very early-stage and ambitious from a technical standpoint. Its set of proposed technologies span too many areas to be compatible with a start-up’s capabilities and resources. In fact, Mobious’ patent portfolio has the breadth one would expect from the likes of Roche Diagnostics or Life Technologies.

Schematic of SPR technology from US 2008/0014592

The following is a sampling of the core components of Mobious’ proposed sequencing methods and patents: 1) detect conformational changes in a single processing enzyme or a change in the polymerase’s mass (e.g. association with a nucleotide) using SPR, TIRM, or other light-based interrogation methods. In one embodiment, nucleotides are added sequentially, and in another, advanced blocking group chemistry is proposed to allow all nucleotide types to be present in the same reaction[2]; 2) measure polymerase dissociation rate from a target strand, leveraging differences in polymerase dissociation rate which are dependent on the presence or absence of a complementary base[3]; 3) detect conformational changes of an enzyme based on FRET[4]; 4) measure a single polymerase’s dielectric constant in order to detect conformational and/or energy level changes indicative of association with a specific nucleotide type[5]; 5) a variation on US2008/0014592 where a helicase is used to accomplish sequencing[6]; and 6) use of other advanced optical methods to detect enzyme conformational states (recent filing)[7].

At this time, Mobious should narrow its focus to only the most promising of its approaches and applications and open its business model to a variety of commercial partnership structures (if it has not done so already). Although fourth generation sequencing should not be ruled out, proteomics and other life sciences tools are likely a better bet. To access fourth generation sequencing, Mobious needs to be able to demonstrate the essential working components of a sequencing prototype at this time – and there should be clear competitive advantage versus other emerging sequencing technologies. Although many of Mobious’ proposed sequencing methods would be too expensive, time-consuming, or error-prone, there are a few strong approaches in the mix. Finally, it’s worth pointing out the degree of secrecy surrounding the company’s activities: there is a lack of information available on its current sequencing capabilities; and, although it lists University of Exeter Innovation Centre as its headquarters[1], the Centre’s tenant list does not appear to include Mobious for whatever reason[8].

Overview: Norwegian start-up LingVitae is developing a tool to translate biological data of interest into a form that can be more readily detected. It’s using a restriction and ligation enzyme system to cleave two end bases at a time from target DNA fragments of around 4- 40 bases[1], and effectively replace such two base combinations with predetermined, longer sequences of DNA (or DNA bound to labels), which are then concatenated into a new and much longer DNA strand. The process can be thought of as making a genetic binary code, where A, T, G, and C are replaced with, for example, 0-0, 0-1, 1-0, and 1-1 (where 0’s and 1’s correspond to distinct 10-base-long units). The resulting DNA concatemers would be amenable to hybridization with probe oligonucleotides[2]. In addition, a similar overall method could be used to convert protein sequences into nucleic acid sequences for detection[3]. Continue reading →

Overview: Start-up Bionanomatrix is developing nanofluidic device components to uncoil and entrain exceptionally long segments of linearized DNA or other biomolecules in nanochannels for subsequent analysis. For genome study and personalized medicine, the approach would enable local sequence information to be readily placed in its much larger spatial and chromosomal context, a feature lacking in current sequencing products. Investors should focus on whether the technology can be effectively developed and brokered as an open-source platform that is compatible with multiple emerging technologies in the field. Bionanomatrix’s technology is built around nanoscale geometric features such as gradually narrowing channels and nanopillar arrays. The devices are designed for parallel analysis of biomolecules and to be compatible with a variety of interrogation methods and probes[1]. Its nanofluidic technology is being marketed as a platform, and the company is currently collaborating with multiple partners[2].

Analysis: A challenge for Bionanomatrix will be to engineer a true open source platform so the company can execute on this lucrative business model. Accordingly, investors should examine previous and upcoming deal structures, and also verify these deals span sufficiently different technologies. There are a number of ways Bionanomatrix can integrate its technology with others, in some scenarios making it an adjunct part, and in other scenarios making it a more essential part of third and fourth generation sequencing and other biomolecule analysis instruments. There’s reason to be skeptical of its partnership with Complete Genomics, as neither party brings a sophisticated detection technology to the table.

Overview: Intelligent Bio-Systems was founded in 2005 and is based on technology from Jingyue Ju’s lab at the Columbia University Genome Center. The start-up is built around molecular designs for reversible terminators and the corresponding synthetic chemistry. Ju has engineered modified, fluorescently-labeled bases that are compatible with polymerase function and which revert to native bases chemistries in a single step reaction between built-in allyl groups and palladium catalyst[1]. Intelligent Bio-Systems raised $2.4 million in a Series A and around $4 million in a recent Series B. To date, it has received around $4.3 million in grants and reportedly closed a small bridge financing prior to its Series B[2]. The company has undoubtedly struggled given the highly competitive set of similar technologies under development at other companies, many with considerable R&D lead time. As a result, Intelligent Bio-Systems is likely to be acquired for its advanced molecular chemistries.

For additional information on Intelligent Bio-Systems or analysis of related technologies and companies, contact: bruce@schiamberggroup.com

Overview: Start-up Halcyon Molecular is developing a method to sequence nucleic acids using high-atomic-number-labeled bases and electron microscopy. This approach to detection was first proposed by Richard Feynman around 1958. Halycon Molecular is also developing a number of supporting techniques, including use of functionalized needles to stretch and place taut DNA onto substrates for subsequent analysis. The company has around 15 employees and is located in the San Francisco Bay Area.